Depletion of Neutrophils in IL-10−/− Mice Delays Clearance of Gastric Helicobacter Infection and Decreases the Th1 Immune Response to Helicobacter This information is current as of October 1, 2021. Hanan F. Ismail, Pamela Fick, Juan Zhang, Richard G. Lynch and Daniel J. Berg J Immunol 2003; 170:3782-3789; ; doi: 10.4049/jimmunol.170.7.3782 http://www.jimmunol.org/content/170/7/3782 Downloaded from References This article cites 43 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/170/7/3782.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Depletion of Neutrophils in IL-10؊/؊ Mice Delays Clearance of Gastric Helicobacter Infection and Decreases the Th1 Immune Response to Helicobacter1 Hanan F. Ismail,* Pamela Fick,* Juan Zhang,* Richard G. Lynch,† and Daniel J. Berg2* Gastric infection with Helicobacter induces a lymphocyte-rich mucosal inflammation that contains a minor population of neu- trophilic granulocytes. The function of neutrophils in the local immune response to gastric Helicobacter infection remains un- known. To investigate this issue, we conducted experiments in neutrophil-depleted control wild-type (wt) and IL-10؊/؊ mice infected with Helicobacter felis by gastric lavage. Infection of wt mice elicited a mild, focal gastritis and a Helicobacter-specific Th1 immune response. In wt mice Helicobacter colonization of the stomach was persistent and progressively increased during the 29 -days of observation. Infection of IL-10؊/؊ mice with H. felis elicited a severe chronic gastritis and a greatly enhanced Helicobacter ؊/؊ specific Th1 immune response, as compared with wt mice. After initial colonization, the IL-0 mice completely cleared Heli- Downloaded from .cobacter from the stomach by day 8. The gastric inflammation in wt and IL-10؊/؊ mice contained modest numbers of neutrophils The intensity of gastric inflammation and the extent of Helicobacter colonization were similar in control and in neutrophil-depleted wt mice. In contrast, neutrophil depletion of Helicobacter-infected IL-10؊/؊ mice decreased the severity of gastritis, modulated the Helicobacter-specific Th1 immune response, and delayed the clearance of bacteria from the stomach. These studies identify a role ,for neutrophils in the local and systemic immune response to gastric Helicobacter in IL-10؊/؊ mice. The Journal of Immunology 2003, 170: 3782–3789. http://www.jimmunol.org/ elicobacter pylori is a human gastric pathogen that can product of neutrophil activation, may also play a role in the patho- cause gastritis, peptic ulcer disease, and gastric cancer. genesis of the disease (12). H The organism was first associated with peptic ulcer dis- Because of the difficulty of performing invasive studies in hu- ease in 1984 (1), and since this time there has been an exponential mans, much of our understanding of the immune basis of H. py- increase in our knowledge of the role of Helicobacter in disease. lori-related disease comes from studies in animal models. Like However, despite advances in the understanding of the biology of humans infected with H. pylori, mice infected with H. pylori or the H. pylori, the factors that determine the outcome of the infection in closely related Helicobacter felis (13) respond to gastric infection by guest on October 1, 2021 an individual host remain poorly understood. with infiltration of Th1-biased lymphocytes into the gastric mu- In recent years, evidence has accumulated to suggest that in both cosa and spleen (14). IFN-␥ becomes elevated in tissue and splenic human patients and animal models, the host cellular immune re- Th cells respond in vitro to H. pylori by secreting high levels of sponse is an important determinant of the outcome of the infection. IFN-␥ (15). IL-10, a potent anti-inflammatory and immune-regu- Indeed, H. pylori-infected individuals express proinflammatory cy- latory cytokine, is also produced during H. pylori infection (16) tokines in their gastric mucosa, and it has been shown that Heli- ϩ and may down-regulate the host response to gastric Helicobacter cobacter induce a Th1-type CD4 T cell immune response (2, 3). infection. H. felis infection in mice with a targeted disruption of In addition to the T cell infiltrates, Helicobacter infection also Ϫ/Ϫ induces a prominent neutrophilic infiltration as well, and it has the IL-10 gene (IL10 ) results in severe inflammation with been proposed that H. pylori-induced damage is related to neutro- metaplastic and dysplastic epithelial changes that occur within 4 phil-mediated tissue injury. In support of this connection is the wk of infection (17). Helicobacter-induced epithelial changes may 3 observation that the extent of mucosal injury is related to the de- take up to 1–2 years to develop in H. felis-infected wild-type (wt) gree of H. pylori infection and neutrophil infiltration (4, 5). Fur- mice (18) and similar changes also occur in some patients chron- thermore, extracts of H. pylori contain substances that elicit che- ically infected with H. pylori, usually over a period of decades. motactic activity in neutrophils and monocytes (6–11). In addition, In this study, we investigated the role of endogenous IL-10 on it has been hypothesized that reactive oxygen intermediates, a the development of a productive immune response to gastric H. felis infection. We observed that both wt and IL-10Ϫ/Ϫ mice de- veloped a Th1-type T cell response to H. felis infection. However, † Departments of *Internal Medicine and Pathology, University of Iowa College of the local and systemic immune response to Helicobacter was much Medicine, Iowa City, Iowa 52242 greater in the absence of IL-10 and was associated with the erad- Received for publication September 5, 2002. Accepted for publication January Ϫ/Ϫ 23, 2003. ication of gastric Helicobacter infection in the IL-10 mice. Our The costs of publication of this article were defrayed in part by the payment of page studies also show that the development of the anti-Helicobacter charges. This article must therefore be hereby marked advertisement in accordance immune response and clearance of Helicobacter from the stomach with 18 U.S.C. Section 1734 solely to indicate this fact. are diminished in the absence of neutrophils. 1 This study was supported by National Institutes of Health Grant R29 CA76129 (to D.J.B.). 2 Address correspondence and reprint requests to Dr. Daniel J. Berg, Department of Internal Medicine, University of Iowa Hospitals, C32-GH, 200 Hawkins Drive, Iowa City, Iowa 52242. E-mail address: [email protected] 3 Abbreviations used in this paper: wt, wild type; ROS, reactive oxygen species. Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 3783 Materials and Methods dinal cross-sections of the stomach from each wt or IL-10Ϫ/Ϫ mouse were Mice examined. The number of infected glands and number of bacteria in each gland were counted in each section to determine the number of bacteria per Healthy 6-wk-old IL-10Ϫ/Ϫ mice on a 129/EvSv background were used for section. The data presented are the mean number for all mice per group, this study (19). Wild-type 129/SvEv mice were purchased from Taconic using the value from the section with the highest number of bacteria. At Farms (Germantown, NY). Mice were maintained in microisolator cages later time points (1 wk and greater for wt mice, 1-wk time point for neu- Ϫ Ϫ under specific pathogen-free conditions at the animal care facility at the trophil-depleted IL-10 / mice), gastric colonization was uniform and bac- University of Iowa (Iowa City, IA). All mice were maintained in accor- terial counts did not vary significantly between sections. dance with guidelines of the University of Iowa Animal Care and Use To further assess colonization, stomach DNA from infected wt and IL- Ϫ Ϫ Committee. 10 / mice was prepared using DNAzol (Life Technologies, Grand Island, NY) exactly per the manufacturer’s instructions. The presence of bacteria Bacteria was assessed via PCR amplification of the 16S ribosomal RNA gene using Helicobacter-specific primers (21). H. felis (ATCC 49179) was obtained from American Type Culture Col- For the reisolation of H. felis, the gastric tissue was rubbed over the lection (Manassas, VA). Bacteria were grown as described previously (20). surface of Brucella agar plates with TVP (Remel) and the plates were Briefly, bacteria were grown on Brucella agar plates with TVP (Remel, cultured under microaerophilic conditions at 37°C. Bacteria were posi- Lenexa, KS) under microaerophilic conditions at 37°C for 2 days. Con- tively identified on the basis of morphology and presence of urease enzyme fluent plates of bacteria were harvested and the number of bacteria was activity. 9 determined by absorption at A450, with 1 OD unit corresponding to 10 bacteria. Bacteria were positively identified on the basis of morphology and presence of urease enzyme activity and via PCR amplification of the 16S Histologic analysis ribosomal RNA gene using Helicobacter-specific primers (21).